Engineering and expression of sequence-specific DNA-binding zinc finger protein / 生物医学工程学杂志

This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.

Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix / 生物医学工程学杂志

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.

Study on the biomechanical behavior of human intestine / 生物医学工程学杂志

To investigate the biomechanical behavior of human intestines. The tensile test human intestine was performed with the electronic tension machine in this paper. The results indicate that the exponential relationship for the stress-strain of the human intestine was obtained, and the exponential coefficient a of each segment of the intestine is almost the same although the constant C is different. It also shows that the relative rate of stretch length of each segment intestines is different in longitudinal and circumferential directions. And the incremental elastic modulus of colon is less than those of small intestine. It is considered that the colon can be more easily deformed. The experimental results provide the theoretic basis for research on intestinal endoscopic microrobot.

Overexpression of TGFbeta1 increases ECM expression and adhesion of endothelial cells on matrix / 生物医学工程学杂志

This study was aimed to examine the effectiveness of a gene transfer of human TGFbeta1 gene into endothelial cells and to determine whether TGFbeta1 increases ECM expression of endothelial cells. With the help of DOTAP, endothelial cells were transfected with pMAMneoTGFbeta1. The positive cell clones were selected with G418. The stable transfection and expression of TGFbeta1 in the endothelial cells were determined by immunofluorescence analysis. The expression levels of collagen I and fibronectin in the transfected and untransfected endothelial cells were determined by Western blot. The adhesion force between endothelial cells and matrix was determined by a micropipette technical system. The results showed abundant TGFbeta1 stable expression in the endothelial cells. TGFbeta1 gene was noted to increase collagen I and fibronectin expression and increase the adhesion between endothelial cells and matrix. These findings indicated that TGFbeta1 can be used in vascular tissue engineering for the enhancement of endothelial cells adhesion.

Study of human smooth muscle cells seeding in biologic blood vessel matrix / 生物医学工程学杂志

To explore the human smooth muscle cells seeding in blood vessel of minor pig after trypsin treatment and provide data for xenotransplantation and for using pig vessel in tissue engineering. HE and silver stain were used for checking the smooth muscle cells seeding in acellular blood vessel. The results showed that the smooth muscle cells seeding succeeded and the smooth muscle cells were in normal morphological distribution. These demonstrate that the pig aorta can be used for smooth muscle cells seeding, and hence for constructing new vascular grafts.

Anatomical research of peritrochanteric femur and clinical application of internal fixation for femoral intertrochanteric fractures / 中华创伤骨科杂志

Objective To measure the proximal femoral parameters which can provide anatomic evidence for the design of internal fixation components for intertrochanteric fractures of femur.Methods Femoral speci- mens were harvested randomly from 120 healthy adult cadavers(left 60,fight 60).The neck shaft angle,greater trochanter slope length,tilt angle of greater trochanter,axial length of head and neck,lengths of upper and lower borders of the neck,and the minimum transverse diameter of the neck were measured.On the basis of the anatomic study,a two-claw plate was designed to treat 145 cases of femoral intertrochanteric fractures.Results The femoral neck shaft angle was 128.59??6.31?,femoral greater trochanter slope length was(5.5?0.58)cm,tilt angle of femoral greater trochanter was 42.76??5.20?,and axial length of head and neck was(9.42?0.38)cm. There was a correlation between the parameters.All the patients were followed up for a mean time of 23.6 months. The fractures got clinic union in 3 to 6 months.Two cases experienced detachment of claws and hooks but their final outcome was not affected.Three cases suffered coxa vara.All the other cases obtained normal motion function of hips and normal neck shaft angle.No breakage of claw,hook or nail was found in them.Conclusions It is necessary to design an internal fixator that can fit the anatomical features of Chinese femurs in the treatment of intertrochanteric fractures of femur.The two-claw plate designed by us is a good attempt to improve the clinical effect.

Antigen expression and the endothelization of biologic blood vessel matrix / 生物医学工程学杂志

This study sought to explore the change of the major histocompatibility complex (MHC) antigen expression and the endothelization of blood vessel in minor pig after trypsin treatment, and to provide data for xenotransplantation and pig vessel for use in tissue engineering. Western blot assays were conducted for detecting the expression of MHC xenoantigens. Scanning electron microscopy was used for checking the endothelization of decellularized blood vessel. The results showed that MHC antigen is not expressed after trypsin treatment. The endothelization is accomplished. The endothelial cells have normal morphological distribution. These demonstrate that the antigen of pig aorta is significantly decreased and it can be used for constructing new vascular grafts.

Zinc finger protein gene A20 inhibits lipopolysaccharide-induced IL-8 expression of endothelial cells / 第三军医大学学报

Objective To investigate the effects of zinc finger protein gene A20 on the inhibition of lipopolysaccharide (LPS) induced interleukin 8 (IL 8) expression in endothelial cells. Methods Plasmid pcDNA3.1EHA20 was transfected into human umbilical vein endothelial cells (HUVECs) by DOTAP method. The positive cell clones were screened with G418. The stable transfection and expression of A20 in HUVECs were determined by immunofluorescent analysis. IL 8 expression was detected by sandwich ELISA with double monoclonal antibodies. Results High expression of A20 gene in HUVECs transfected with pCDNA3.1EHA20 was confirmed by immunofluorescent analysis. IL 8 expression increased in LPS induced endothelial cells. A20 gene could inhibit more than 70% LPS induced IL 8 expression ( P

Construction and identification of recombinant adenovirus shuttle plasmid pAdCMVLacZ / 第三军医大学学报

Objective To construct replication deficient adenovirus vector AdCMV lacZ Methods Recombinant adenovirus shuttle plasmid pAdCMVlacZ was first constructed and then lacZ cDNA cloned into Hind Ⅲ and Eco R Ⅴ of adenovirus shuttle plasmid pAdCMV by conventional methods Results The restriction enzymatic map of pAdCMVlacZ accorded with theoretic analyses and pAdCMVlacZ showed blue bacterium groups under the existence of revulsant and X gal Conclusion The adenovirus shuttle plasmid pAdCMVlacZ has been constructed successfully

Overexpression of TGF beta 1 increases elastin expression and adhesion of smooth muscle cells / 生物医学工程学杂志

This study was conducted to examine the effectiveness of a gene transfer of human TGF beta 1 gene into smooth muscle cells and whether the TGF beta 1 can increase elastin expression of smooth muscle cells. With the help of DOTAP, smooth muscle cells were transfected with pMAMneoTGF beta 1. The positive cell clones were selected with G418. The stable transfection and expression of TGF beta 1 in the smooth muscle cells were determined by immunofluorescence analysis. The expression of elastin in the transfected and untransfected cells were determined by in situ hybridization. The adhesion force between smooth muscle cells and matrix was detected by micropipette system. The results showed abundant TGF beta 1 stable expression in smooth muscle cells. TGF beta 1 gene can increase two-three times elastin expression and increase the adhesion between smooth muscle cells and matrix. TGF beta 1 can be used in vascular tissue engineering to increase smooth muscle cells adhesion.

Synthesis of a triple helix-forming phosphorothioate oligodeoxynucleotides and its effects on coagulation activity of tissue factor (TF) and TF gene expression in endothelial cells / 生物医学工程学杂志

This study sought to synthesize a triple helix-forming phosphorothioate oligodeoxynucleotides (TFO-ps) and assess its effects on coagulation activity of the tissue factor(TF) and TF gene expression in endothelial cells. Experiment antiparallel oligodeoxynucleotides T21GTa sequence was designed and synthesized by phosphoramidite method and decorated with all-PS linkage. The affinity of TFO and TFO-ps was determined by electrophoretic mobility shift assays(EMSA). Cellular uptake of [32]P-labeled TFO-ps and the effect of TFO-ps on TF gene expression and coagulation activity of TF were measured in endothelial cell strain ECV304. The results showed that TFO-ps (T21GTa-ps) formed a triplex binding in antiparallel orientation to the puring-rich target strand-SSRE, with Kd value of 3.6 x 10(-10) M. The uptake rate of TFO-ps (T21GTa-ps) in ECV304 was about 11.65%. This compound mainly localized within the nucleus sediment(77.25%), significantly reduced the average OD of the mRNA expression and protein synthesis of TF gene, and obviously decreased the coagulation activity of TF. In conclusion, TFO-ps (T21GTa-ps) shows obvious anti-coagulation activity and its mechanism involves the inhibition of TF gene expression.

Application of four-dimensional mathematic model in cerebral edema in early stage after severe burn in dogs / 中华创伤杂志

Objective To explore the spatio-temporal regularity of cerebral edema in the early stage of severe burn (50% TBSA Grade Ⅲ) with four-dimensional (4D) mathematic model. Methods Twenty-six dogs were randomly divided into control group and 6, 12, 18 and 24 hours post-burn groups. The dogs in the postburn groups suffered severe burn of 50% TBSA GradeⅢ. Six hours after burn, 50 g/L glucose was injected according to Parkland formula. MRI manifestation, histopathological changes, brain water content and intracranial pressure were observed in each group. A 4D mathematic model was established based on the results of MRI scanning. Results Two turning points ( 6 and 18 hours postburn) and three phases of pathologic changes were displayed by 4D mathematic model of cerebral edema in the early stage of severe burn. The first phase (6 hours postburn) was in the subclinical period,when an effective treatment should be taken as quickly as possible so as to prevent deterioration of postburn cerebral edema. The second phase ( 6-18 hours postburn) was in the apostatic period with pathologic characteristic of vasogenic and cytotoxic brain edema. The third stage (18-24 hours postburn) was the critical period of cerebral edema. Intracerebral pressure (ICP) increased rapidly due to limitation of cranial cavity, when cerebral hernia easily happened. Conclusions The 4D model indicates that there is a feature of type S curve in the pathologic process of cerebral edema in the early stage after severe burn.

Effect of adenovirus-mediated bcl-2 transfer on regeneration and repair of peripheral nerves in rats / 中华创伤杂志

Objective To explore the effect of adenovirus-mediated bcl-2 transfer on the regeneration and repair of peripheral nerves in rats. Methods The sciatic nerve-transected model was selected and sutured end to end with epineurium. Then, adenovirus/sense-B-lymphoma/leukemia-2 (Ad/s-bcl-2), adenovirus/anti-sense-B-lymphoma/leukemia-2 (Ad/as-bcl-2), adenovirus/?-galactosidase (Ad/LacZ) and normal saline (NS) were injected into the sciatic nerve 0.5 cm away from the sutured point respectively. The rats were perfused with 4% paraformaldehyde at the 48th hour, 7th, 15th and 30th days respectively after operation. The spinal cords L 4-6 were harvested, frozen sectioned and given x-gal staining, bcl-2 in situ hybrid and immunohistochemical staining and GAP-43 immunohistochemical staining. The postoperative functional recovery of the rear limbs of rats was dynamically observed by using sciatic nerve functional index (SFI). Results The GAP-43 expression in the motor neurons of anterior horn of spinal cord in group Ad/s-bcl-2 was higher than that in other groups (P0.05). Conclusions Gene transfer of bcl-2 can promote regeneration and functional recovery of the peripheral nerves following sciatic nerve injury.

Construction and identification of mouse Osf2/Cbfal gene recombinant adenovirus / 第三军医大学学报

Objective To construct the recombinant adenovirus of mouse Osf2/Cbfal gene and to observe its ability to infect NIH3T3 fibroblasts. Methods The Osf2/Cbfal gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into NIH3T3 cells using Lipofectine DOTAP, The target gene was detected by poly-merase chain reaction (PCR). The titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. Results Restriction endonuclease and PCR analyses confirmed that the Osf2/Cbfal gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.6?1012 pfu/ml. The adenovirus had a strong effect on NIH3T3 cells. Conclusion The recombinant adenovirus containing Osf2/Cbfal gene was successfully constructed by the method of homogenous recombination in bacteria.

Experimental study of the mouse bone marrow stromal cells differentiated into neural-like cells / 第三军医大学学报

Objective To observe the growth and differentiation of the cultured mouse bone marrow stromal cells in vitro into neural like cells. Methods Bone marrow stromal cells (BMSCs) isolated from mice were cultured according to the routine method, and induced to differentiate by basic fibroblast growth factor b (bFGF), retinoic acid (RA), nerve growth factor (NGF), and dimethylsulfoxide (DMSO). Expressions of neural microfilament protein (NF 200), glial fibrillary acidic protein (GFAP), and fibronectin at 72 h after inductin were determined by immunoflurenscence and immunocytochemicalmethod. Results At 72 h after inductin, neural like changes were found in BMSCs. Immunohistochemical stain showed that 60% BMSCs differentiated into NF 200 positive cells, and about 50% BMSCs into GFAP positive cells. Fibronectin expression was significantly lower as compared with that in the control group. Conclusion Combined action of RA, bFGF, NGF, and DMSO can induce BMSCs to differentiate into neural like cells in vitro .

Antigen expression and the biomechanical characteristics of the biologic blood vessel matrix / 生物医学工程学杂志

To explore the changes of the antigen expression and the biomechanical characteristics of blood vessel in Banna little ear pig before and after trypsin treatment, and provide data for xenotransplantation and pig vessel using for tissue engineering. Geometric morphology and microstructure of pig cartoid artery were stuided quantitatively by histologic method and computer image analysis. The relationship between pressure and diameter was observed at different period of time before and after trypsin treatment. Affinity-immunohistochemistry assay was conducted to detect the expression of xenoantigens (alpha-Gal). The results showed that alpha-Gal antigen is only expressed in vascular endothelial cellsouly. There is no significant difference in blood vessel compliance. These demonstrate that the antigenicity of pig carotid artery is significantly reduced, however, the mechanical characteristics did not change significantly. We suppose that pig vessels treated by trypsin can be used as the substrate material for vascular tissue engineering.

Influence of flow shear stress on apoptosis of arterial endothelium in vivo / 生物医学工程学杂志

To explore the changes of wall shear stress(WSS) effect on arterial endothelial cell(EC) apoptosis after reducing arterial blood flow. The reducing flow model was established in 60 rabbits. Endothelial stretched preparations were made at 8 different time intervals from 0 to 30 days. The apoptosis rate of arterial endothelial cells (AEC) was measured with TdT-mediated dUTP-biotin nick end labeling(TUNEL) method. The results showed that the apoptosis rate of AEC was significantly higher from 1 day to 7 days after decreasing WSS than that of control, which peaked on day 3. While with progressively increasing in WSS, the apoptosis rate restored to the level of control from 14 days to 30 days. These suggest that the apoptosis state of AEC might be markedly influenced by the changes of WSS. The persist decreasing of WSS may be the important factor which induces the cell apoptosis.

OVEREXPRESSION OF ZINC FINGER PROTEIN INHIBITS LPS-INDUCED EXPRESSION OF E-SELECTIN IN ENDOTHELIAL CELLS / 解剖学报

Objective Previous studies have demonstrated that LPS can induce endothelial cell activation and the expression of E-selectin. In this study, we examined whether A20 gene could inhibit the expression of E-selectin in endothelial cells induced by LPS. Methods With the help of DOTAP, endothelial cells were transfected with pCDNA3.1 EHA20. The postive cell clones were selected with G418.The stable transfection and expression of A20 in the endothelial cells were determined by immunofluorescence analysis. The E-selectin expression was checked by immunofluorescence, Western blot and in situ hybridization. Results Abundant A20 stable expression in endothelial cells transfected with pCDNA3.1 EHA20 was confirmed by immunofluorescence analysis. E-selectin expression increased in LPS-inducible endothelial cells. A20 gene inhibited 90% LPS-inducible E-selectin expression(P

CULTIVATION AND OSTEOBLASTIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS ON GFP-EXPRESSING MOUSE IN VITRO / 解剖学报

Objective To isolate the bone marrow mesenchymal stem cells(MSCs) from the GFP-expressing mouse, and to study the osteoblastic differentation of the cells. Methods MSCs were isolated by density gradient centrifugation, then the clutrued cells were induced to osteoblastic differentiation using the conditional medium. We detectd the expression of GFP and MSCs differentiation into osteoblasts by histochemistry and immunochemistry. Results The MSCs maintained the expression of GFP during expanded and induced process. After induced for 10 days, lots of alkaline phosphatase and osteoclcin staining positive cells were observed.Conclusion The MSCs of GFP-expressing mouse were successfully isolated and differentiated into ostoblasts. It may be valuable for tracing the seeding cells in tissue engineering bone.

Biomechanical testing and evaluation of a self-made allograft interbody fusion cage at lumbar spine / 第三军医大学学报

Objective To evaluate the stability and compressive mechanical functions of the lumbar spine following insertion of a new self-made allograft interbody fusion cage.Methods Anti-bending intensity with three points test,anti-rotation intensity and compressive stiffness were measured at L4-L5 lumbar spine on five adult human fresh cadaveric specimens following insertion of a new self-made allograft interbody fusion cage and compared with that of before and after nucleus pulposus removal.Results The anti-bending intensity of flexion and extension of lumbar spine after inserting a new allograft interbody fusion cage was increased significantly(P